Photobiomodulation: A Systematic Review of the Oncologic Safety of Low-Level Light Therapy for Aesthetic Skin Rejuvenation

Abstract   Photobiomodulation (PBM) therapy is an increasingly popular modality for aesthetic skin rejuvenation. PBM induces genomic, proteomic, and metabolomic processes within target cells, but such manipulation of cell behavior has led to concerns about oncologic safety. This article presents a summary of the clinical and preclinical evidence for the oncologic safety of PBM for aesthetic skin rejuvenation. A focused systematic review was performed, in which safety data from clinical trials of PBM for skin rejuvenation was supplemented by analyses of in vitro data obtained from cells derived from human skin and human neoplastic cells and in vivo data of tumors of the skin, oral cavity, and breast. Within established parameters, red and near infrared light mainly enhances proliferation of healthy cells without a clear pattern of influence on cell viability. The same light parameters mainly reduce neoplastic cell proliferation and viability or else make no difference. Invasiveness potential (appraised by cell migration assays and/or differential gene expression) is equivocal. PBM does not induce dysplastic change in healthy cells. In vivo tumor models yield varied results with no clear pattern emerging. There are no relevant clinical trial data linking PBM with any significant adverse events, including the finding of a new or recurrent malignancy. Current clinical and preclinical evidence suggests that PBM is oncologically safe for skin rejuvenation, and there is no evidence to support the proposition that it should be avoided by patients who have previously undergone treatment for cancer. Level of Evidence: 4

The reason for taking this question seriously lies in the complex physiological responses to low-level laser light. Red and near infrared light energy is absorbed by intramitochondrial chromophores including cytochrome c oxidase (CcO) and perhaps one or more flavoenzymes. The absorption of light by CcO (an important enzyme in the electron transport chain) facilitates electron transport and drives adenosine triphosphate (ATP) synthesis. ATP, in turn, drives cell metabolomics. 9 Moreover, in a localized environment of oxidative stress, red and near infrared light dissociates nitric oxide (NO), competitively bound to CcO at the expense of oxygen, restoring functionality to the electron transport chain and releasing NO to induce localized vasodilation, thereby improving localized oxygen delivery and the migration of cells of innate immunity. Simultaneously the absorption of light by flavoenzymes such as flavin dehydrogenase alters the reduction/oxidation (redox) state of the intracellular microenvironment. Not only does the balance of reactive oxygen species influence mitochondrial ATP production as detailed above, it alters the transcriptome to favor the expression of genes implicated in regeneration and repair and, thereby, the recruitment of cells of innate and adaptive immunity implicated in the coordination of tissue repair. [10][11][12][13] A more detailed description of the role of PBM on cell signaling, immune modulation, and regulation of microcirculation is provided elsewhere. 14,15 In a sense we have now come full circle, because the first experiments with low-energy red laser light were actually performed for the purpose of establishing oncologic safety over 50 years go; experiments that yielded incidental evidence of accelerated hair regrowth and enhanced wound healing but, it should be noted, no evidence of neoplastic change. 16,17 With a large body of clinical and experimental evidence now behind us and the popularity of PBM booming, it is time to reexamine the evidence to establish whether this therapy is safe for our aesthetic patients, including those who have previously undergone treatment for cancer or those with an undiagnosed dysplasia or neoplasia, and to enable us to address the queries and concerns of our well-informed and curious patients.

Search Strategy
A literature review was conducted to identify articles pertaining to the oncologic safety of PBM when utilized for aesthetic skin rejuvenation, including scar management. The study was performed by GG and repeated by AH and JB (see acknowledgments). Thus, 3 independent searches were performed. Discrepancies were handled by the sole author (GG), who made the final decisions on source inclusion. The search was conducted with Google Scholar (1997 to May 2022; Google, Mountain View, CA); PubMed (1997 to October 2022; US National Library of Medicine, Bethesda, MD); OVID Medline (January 1997 to October 2022; Wolters Kluwer, Alphen van den Rijn, the Netherlands); and the Cochrane database of systematic reviews and the Cochrane controlled trials register (searched July 18, 2022; Wiley, Hoboken, NJ). A 25-year range was chosen because it was felt that this best encapsulated the contemporary literature on the subject. Both preclinical (experimental) in vitro and in vivo studies and clinical trials were included. The search was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). 18 The search strategy is summarized in Supplemental Table 1 and the PRISMA checklist is provided in Supplemental Table 2.

Inclusion Criteria
All evidence was sought for the oncologic safety of PBM when utilized for skin rejuvenation. In vitro studies were included if they employed human primary cells, stem cells, or tumor cell lines derived from skin, breast, fat, salivary or oral mucosa, endothelium, or hair follicles. These included mesenchymal stem cells, adipocytes, fibroblasts, keratinocytes, melanocytes, Langerhans cells, endothelial cells, ductal epithelial cells, and lobular epithelial cells and the associated precursor cells.
With in vivo studies, investigators address the shortcomings of in vitro study design by examining tumor cell response in the context of tissue architecture and host immune response. By their nature, most in vivo studies use animal models and cell lines. However, when the cell line and in vivo model afforded the opportunity of a direct comparison with a neoplastic process of clinical relevance to the use of PBM, the inclusion of such studies was thought to be relevant. In vivo studies of relevance to our investigation included oral squamous cell carcinoma (and other tumors of the head and neck); melanoma; and breast (adeno)carcinoma. The fact that this was the only section containing animal data meant that it could be evaluated on its own merits without clouding the overall picture.
Clinical trials were included if the data they contained related to the oncologic safety of PBM or could be extrapolated to draw inferences about oncologic safety when employed for skin rejuvenation. Only PBM applied with light of wavelengths at the red (625-700 nm) and infrared (700-1100 nm) end of the spectrum were included, because this represented the spectrum of light utilized in PBM for skin rejuvenation commercially.

Exclusion Criteria
In vitro studies utilizing nonhuman cells and nonhuman (veterinarian) clinical trials were excluded. Similarly, in vitro and in vivo studies using primary cells or tumor cell lines Glass NP359 with no direct or indirect correlation to skin rejuvenation (eg, diabetic fibroblasts and keratinocytes, bone marrowderived stem cells, endothelial cells) and nonrelevant clinical studies (eg, PBM for radiation mucositis) were excluded. Studies that did correlate with skin rejuvenation but reported only on the efficacy of PBM and not on oncologic safety were also excluded because they have been considered elsewhere. 1 Only experimental data were considered; therefore reviews were excluded from the analyses. Thermal light exposures, including high-energy laser, intermittent pulsed light, and photodynamic therapy, as well as PBM with light of wavelengths other than those in the red/ near infrared end of the spectrum were also excluded. Low quality clinical trials, letters, or short reports that relied exclusively on subjective data were not considered.

Data Extraction
In accordance with the recommendations of Jenkins and Carroll (2011) on the minimum reporting standards for PBM, the following irradiation parameters were noted when available: source of light, wavelength, power, beam area, duration of exposure (therefore, by calculation, fluence), and continuous or pulsed wave, as well as the characteristics of the study including participants, therapeutic protocol, objective outcome measures, and findings. 19 The nature of the clinical safety data or the experimental data on which safety conclusions could be drawn was noted.

Data Analysis
Following extraction of the raw data, analysis revealed a large number of variables with respect to both light and experimental model. These included light source, wavelength, power, fluence (energy per unit area), and exposure protocol; and model cell type and source, species, culture conditions including the presence of additional experimental variables, experimental protocol, and methods of evaluation of effect.
For this reason, it was felt that an attempt to perform quantitative analysis would achieve little in terms of clarity. Although systematic review methodology was observed, synthesis of the evidence took the form of a narrative review. The narrative synthesis was constructed with a specific focus on the need of the aesthetic practitioner to understand the evidence for the oncologic safety of PBM in clinical practice.

RESULTS
A total of 57 papers were included for analysis, comprising 41 in vitro studies, 9 in vivo studies, and 7 clinical trials.  Study selection is shown in Figure 1.

The Influence of PBM on Normal Human Cell Behavior in Culture
The features of normal human cell behavior in culture that might be altered by PBM include proliferation, migration, differentiation, and viability. The present research identified 18 relevant in vitro studies in the last 25 years that utilized primary human cells or cell lines of interest in the context of skin rejuvenation; in which PBM was investigated by exposing the cells in culture to monochromatic light with or without other alterations in the culture protocols; and which examined one or more of these 4 cellular responses. The important light variables included light source, wavelength, total energy density per exposure, and number of exposures. The cells utilized were most commonly fibroblasts and keratinocytes. The culture conditions sometimes included the addition of pharmacologic or physiologic agents to the culture medium to permit examination of the influence of PBM in the presence of another variable.
Seventeen of 18 studies employed laser light, and a single study utilized LED light. Wavelengths of 450 to 880 nm were examined, although most studies used laser light at between 630 and 660 nm. In 13 of 18 studies enhanced cell proliferation under at least one PBM parameter was observed. In the 3 studies in which diminished cell proliferation in at least one PBM parameter was observed, 1 was the study employing LED light at 450 and 530 nm (wavelengths above 530 nm appeared to exert no effect on cell proliferation), 1 utilized infrared laser light (880 nm), and in the third diminished cell proliferation with a highenergy density (≥10J/cm 2 ) of red light (632.8 nm) was observed. Two of the 3 studies in which migration was evaluated reported enhanced cell migration within a narrow energy density of 0.5-5J/cm 2 . Of the 13 studies in which cell viability was evaluated, 6 described changes in cell viability associated with PBM, either in isolation or in the context of a second experimental variable, whereas no change was observed in 7. A summary of the evidence for the effects of PBM on human cells in culture is presented in Table 1.

The Influence of PBM on Neoplastic Human Cell Behavior in Culture
In the context of PBM, relevant neoplastic cell behaviors that can be investigated in vitro include cell viability, proliferation, and invasiveness potential, which include cell migration and the expression of gene products that might serve to confer invasiveness. A total of 23 relevant studies with 34 relevant cell types were identified that met the search criteria. In these studies, 16 head and neck (including oral) squamous cell carcinoma cell lines, 10 breast adenocarcinoma or ductal carcinoma cell lines, 6 melanoma cell lines, and 2 laryngeal carcinoma cell lines were investigated. In four studies, LED light sources were employed, and the remainder used lasers. Although blue (410 nm) and infrared (1064 nm) light were investigated as part of the protocols of some studies, in most studies the effects of low-level red and near infrared light were examined, as was done in the in vitro studies on normal human cells.
In 16 studies with 25 cell lines, the influence of PBM on neoplastic cell viability was examined. PBM was found to reduce the viability of 13 of 25 neoplastic cell lines either in isolation or in the presence of another experimental agent, and PBM was found not to influence the viability of a further 8 cell lines. Of the remaining 4 cell lines, the viability of 3 was found to be dependent on total energy per unit area (fluence), whereas the viability of the remaining cell line was dependent on wavelength of light, exhibiting reduced viability when exposed to blue light only.
In 14 studies with 21 cell lines, the effect of PBM on neoplastic cell proliferation was investigated. The results were mixed. PBM suppressed proliferation in 5 experimental protocols and made no observable difference in another 5. PBM enhanced cell proliferation in 3 experimental protocols. In the remaining 8 experimental protocols, the effect of PBM was observed to vary depending on the experimental parameters, particularly total energy density (fluence) on 5 occasions. In single experimental protocols, cellular proliferation was noted to be dependent on total energy (number of light exposures), wavelength of light, and a mix of wavelength and fluence respectively.
In 4 studies with 4 cell lines, the effect of PBM on the invasiveness potential of neoplastic cells was examined; 3 by way of cell migration assays and 1 by way of investigating differential gene expression. In 2 studies it was concluded that PBM reduced neoplastic cell migration; specifically, red and near infrared light in two studies and blue light in the third. In one study red light appeared to enhance cell migration but the energy density utilized to achieve this result was unclear. In the remaining study, red light (660 nm) was associated with increased expression of MMP-9 and reduced expression of E-cadherin which, together, increase the potential for cell mobilization within the extracellular matrix. A summary of the evidence for the effects of PBM on neoplastic cells in culture is presented in Table 2.

Evidence for Pro-oncogenic Effects of PBM on Normal Human Cells in Culture
Having evaluated the influence of PBM on normal and neoplastic human cells, evidence was sought to establish whether PBM has been implicated in dysplastic or neoplastic change in healthy human cell lines. No human cell in vitro experimental evidence was found to support this view.

The Influence of PBM on (Solid) Tumor
Evidence was next sought for the effect of PBM on established tumors. By definition, it was not possible to restrict this part of the investigation to human tissue alone, and therefore all animal models were considered, as long as it was possible to conceive that the tumor under investigation might theoretically be stimulated by cutaneous light exposure. Hence, visceral tumors and non-solid-state tumors were excluded. A total of 10 relevant models were described in 9 papers. Three models utilized nude mice as the host species for human tumors, 4 were mouse tumors, 2 were hamster tumors, and 1 was a tumor in a rat. The tumor investigated was squamous cell carcinoma in 5 models (oral in 4; skin in 1), breast carcinoma in 2, melanoma in 2, and thyroid carcinoma in the remaining model. Laser-based light was employed in 8 of 10 models, with LED-based light used in the remaining 2. Again, red and/ or near infrared light was utilized in all cases, whether or not other wavelengths were also part of the investigative protocol. Four models in 3 studies also introduced additional variables (radiotherapy, imiquimod, interferon-α receptor knockout mice), which were not considered in detail. The tumor characteristic studied included tumor growth in 6, tumor grade in 2, and tumor gene expression in 1. The remaining model was utilized to study host survival. Of the 6 studies in which tumor growth was investigated, in 3 it was concluded that PBM with red and/or near infrared light did not influence tumor growth, whereas in 1 the conclusion was that PBM inhibited tumor growth (a second study reported that blue light at 405 nm inhibited tumor growth but red light did not). In 2 studies it was found that PBM increased tumor growth. In one study, immunocompromised mice were the hosts for a human tumor and in the other, the energy density associated with tumor growth was in excess of 1000J/cm 2 , which, being very much higher than in any of the other studies, was an outlier in light parameters.
Of the 2 studies in which tumor grade was examined, the conclusion in 1 was that PBM increased tumor grade, and in the other it was concluded that it made no difference. In the study in which tumor gene expression was explored, it was observed that PBM was implicated in the expression of genes associated with cell death through the synthesis of reactive oxygen species. In the study in which host species survival was considered, PBM made no difference. The evidence for the influence of PBM on solid tumor obtained with in vivo preclinical models is summarized in Table 3.

The Influence of PBM on Normal Tissue
No evidence was found to associate PBM with neoplastic change in healthy tissue.

Clinical Trials
In the clinical arena, PBM has been adopted extensively for the management of skin and mucous membrane irritation following radiotherapy for head and neck and breast cancers without evidence of pro-oncogenic effects. [75][76][77][78] PBM is also utilized for skin rejuvenation, subcutaneous lipolysis, hair loss, and the management of pain and sensitivity associated with dental bleaching. To confirm safety, including oncologic safety of PBM for clinical aesthetic use, adverse event reports from clinical trials were sought.

Adverse Event Reporting in Clinical Trials of PBM for Aesthetic Use
The efficacy and safety of PBM for subcutaneous fat reduction, the treatment of alopecia, and pain associated with dental bleaching have been the subject of recent systematic reviews. No evidence was found to support the view that PBM was clinically harmful in general or pro-oncogenic in particular in any of these review studies. [79][80][81] Seven clinical trials of PBM for skin rejuvenation were identified. Mild, transient erythema was reported in a total of 4 patients. No other adverse events were reported in any of these trials. These findings are summarized in Table 4.

DISCUSSION
Photobiomodulation enhances mitochondrial ATP production and alters the reduction oxidation state of the intracellular microenvironment, features which alter the behavior of the cell. 15 An enhanced understanding of the physiologic mechanisms underpinning PBM has given rise to the exploitation of light for clinical therapeutic and commercial benefit. However, theoretic concerns about the oncologic safety of PBM deserve to be addressed. 82 Moreover, the myriad of variables, including light source, wavelength,   but in this case, to answer the question more comprehensively, the decision was made to then work backward, sequentially, to studies of the effect of PBM on solid tumors in an experimental setting and from there the effect of PBM on neoplastic, dysplastic, and finally healthy human cells. The findings of the endeavor were as interesting as they were, in some ways, unexpected. The finding that PBM potentiates the proliferation, viability, and migratory potential of primary human cells, at least within established parameters of wavelength and energy density, was predictable, given that PBM is exploited for aesthetic skin rejuvenation, wound healing, and scar management. 83 The variations in experimental protocols provide ample room to explain differences in observed responses to PBM. A number of light parameters appear crucially important in the efficacy of PBM, and variations in these parameters may account for variations in clinical activity. Specifically, because the chromophore is bound to cellular mitochondria, it has been observed that cells with higher numbers of mitochondria (such as skeletal muscle) tend to respond better to lower doses of light, whereas cells with lower numbers of mitochondria (such as fibroblasts and keratinocytes) require higher doses. 3 What was more surprising were the findings that PBM either reduced or at least appeared to have no influence in most experimental protocols designed to study neoplastic cell viability, and reduced or appeared to have no influence on half of experimental protocols designed to study neoplastic cell proliferation and migration respectively. It is true that these experimental protocols were, in some cases, different in that PBM was investigated in the presence of a second variable in 6 of the 24 experimental protocols. Nevertheless, PBM was implicated in reactive oxygen species-induced cell damage and apoptosis in several different experimental protocols. Additionally, no experimental evidence was found to suggest that PBM can induce neoplastic change in healthy cells. Although it is easy to overlook this negative finding, this is nonetheless important. Following exposure to LLLT, healthy cells stay healthy. The equivocal findings of this part of the study echo those of a recent review that looked specifically at the tumor modulating effects of PBM on head and neck squamous cell carcinoma. The author of this study concluded that the evidence failed to support a clear conclusion. 84 Frankly, it is important not to read too much into in vitro culture models. 85 Crucially, they do not adequately represent the relatively greater complexity of the in vivo tissue microenvironment, a dynamic system characterized by complex interactions between different cells based on molecular cross signals coordinated by cells of innate and adaptive immunity. A total of 10 experimental protocols in 9 papers examined the influence of PBM on solid tumors in a range of animal models. The results require careful consideration. In brief, 4 of 6 models that examined tumor growth concluded that PBM either suppressed tumor growth or, more commonly, made no difference. The 2 experimental protocols that observed an increase in tumor growth in response to PBM might reasonably be considered nonrepresentative. In one case, the energy density was over 1000J/cm 2 , which is very much higher than the energy density typically defined for low-level light therapy. The other, by employing nude mice to host human tumors, eliminated the crucial role of host immune surveillance in the biological response to tumor. This was 1 of 3 experimental protocols that employed nude mice. Of the other 2, in 1 study no difference was observed in tumor survivability using PBM, and in the other PBM did not attenuate radiotherapy-induced tumor shrinkage. Among immunenormal in vivo models, the evidence synthesized in this study supports the conclusion that, with established parameters, PBM probably does not enhance tumor growth or virulence, nor does it induce cancer in normal tissue.
Previously published reviews of clinical trials have concluded that PBM may be useful in attenuating some of the collateral damage of oncologic therapy without diminishing the efficacy of the oncologic therapy. [76][77][78] In fact, it may even play a supportive antitumor role. 7 It would appear that it does so by priming the cellular response to oxidative stress, helping to mitigate the destructive effects of reactive oxygen species induced by radiation or chemotherapy. 86,87 That said, it would be wrong to assume that the role of PBM in oncologic management is limited solely to the management of the side effect profiles of standard oncologic therapy. Near infrared light is currently attracting interest for utilization with lightactivated nanoparticle dispersal systems for precision therapeutic delivery in the management of melanoma. 88,89 The abundance of evidence for the utilization of PBM as a means of managing the side effects of chemoradiotherapy, including oral mucositis, radiation dermatitis, and lymphedema, is a separate issue, and it would be unwise to draw conclusions about oncologic safety based on the findings that PBM can be effective in the management of the side effects of cancer therapy. Nevertheless, a recent systematic review of all of the clinical and preclinical evidence reached a similar conclusion to the present focused study. 90 Moreover, recent systematic reviews on the use of PBM for the enhancement of osseointegration of dental implants and pain associated with dental analgesia yielded mixed results in terms of efficacy but, again, no evidence of harmful side effects. 91,92 However, as in the case of aesthetic practice, an accurate appraisal of the role of PBM in clinical dentistry is noted to be hampered by inconsistencies in the PBM parameters utilized. 93,94 The alternative way to review the findings of this study is that the quality of clinical evidence is simply not good enough to be able to accurately define the risks and limitations of PBM. Although this might be true, PBM has now become so widespread that it might reasonably be anticipated that a harmful incident would have come to light if PBM had the potential to cause harm. Additionally, it is worth mentioning again that the earliest studies of PBM were looking for evidence of harm and did not find any. What this study adds to the existing knowledge base is confirmation of the lack of adverse events reported in clinical trials and case series of PBM for aesthetic use, and a comprehensive summary of in vitro data from human cells that largely supports the conclusion that primary and neoplastic cells behave differently when exposed to low-level light radiation.
There are a number of limitations to this study. Most of the clinical evidence of efficacy and safety in aesthetic clinical practice comes from methodologically flawed, singlecenter clinical trials and case series. Well-designed, large scale multicenter randomized controlled trials (RCT) of efficacy and safety would be very welcome, but the likelihood is remote at present. There may be cause for optimism that, with the accumulation of evidence from small clinical trials, sufficient momentum may be gained to support funding for an RCT. The number of parameters involved continues to make interpretation of the results challenging. It is for this reason that this review and others have adhered to a narrative review structure, without employing the tools that define high quality systematic reviews of clinical trials such as a risk of bias, heterogeneity assessments, and sensitivity analyses. For the same reason, this review does not employ any statistical analytic tools, because there are too many experimental variables to accommodate within quantitative analytic models for any conclusions drawn to be statistically meaningful.

CONCLUSIONS
PBM is increasingly being utilized at home to supplement office-based surgical and nonsurgical aesthetics. It is important that we are in a position to counsel our patients wisely, and to do so in the context of PBM in a patient who has previously had cancer places a special demand on us to understand the evidence. Without a means of navigating the disparate strands of evidence, clinicians may feel uncomfortable about providing an opinion. Despite theoretical concerns there is no clinical evidence to support the proposition that PBM should be avoided in patients who are being treated for cancer, previously had cancer, or have risk factors for cancer. Care must be taken not to read too much into in vitro and in vivo studies. Nevertheless, an evaluation of these data suggests that the behavior of neoplastic cells and solid tumor in response to PBM differs from the behavior of healthy cells and is much more likely to be, although not always, inhibitory. Although clinical advice is always based on evidence that may be subject to revision over time as the body of evidence evolves, the reasonable conclusion from the evidence we currently have at our disposal is that PBM is safe for aesthetic purposes, including for patients with an oncologic history.

Supplemental Material
This article contains supplemental material located online at www.aestheticsurgeryjournal.com.